This application addresses broad Challenge Area (14) Stem Cells and specific Challenge Topic, 15-HL-102: Develop new therapeutic strategies for heart, lung, and blood diseases based on microRNA technology. Congenital heart malformations are the most common birth defects. Mutations in TBX5, a member of the T-box transcription factor family, cause Holt-Oram syndrome (HOS), a heritable disorder characterized by cardiac and upper limb malformations. The heart is exquisitely sensitive to T-box gene dosage, and small perturbations in TBX5 expression have significant cardiac effects. MicroRNAs (miRNAs) are endogenous ~22 nucleotides RNAs that regulate gene expression at the post-transcriptional level by binding the 3'UTR of target genes. They fine tune target genes'expression and regulate many biological processes, including heart development. Our preliminary data show that let-7 miRNA can regulate TBX5 expression by binding to a highly conserved target site in the TBX5 3'UTR. We have identified let-7 mature miRNA expression in both chick and mouse embryonic hearts and in the chick proepicardial organ (PEO). Moreover, we have shown that inhibition of let-7 in chick cultured cardiomyocytes leads to increased Nppa and Gja5 expression, a signature of increased TBX5 activity. Based on these data, we hypothesize that let-7 plays a critical role in cardiogenesis through the regulation of TBX5 expression in both the myocardium and the PEO. Thus, we propose (1) To define spatiotemporal patterns of mature let-7 cardiac expression during vertebrate heart development;(2) To identify the effect of let-7 modifications on TBX5 function in both the myocardium and the proepicardial organ, and (3) To determine the consequences of let-7 knockdown on mammalian cardiogenesis. To achieve these aims we will: (1) perform let-7 in situ hybridization during chick and mouse heart development and employ a "sensor" transgene to study let-7 expression and activity during mouse cardiogenesis;(2) use somatic transgenesis in chick to overexpress and downregulate let-7 in the heart and PEO and will measure cell proliferation and Nppa and Gja5 expression as markers of TBX5 activity, and (3) employ a Cre driver-based system to conditionally downregulate let-7 in a lineage dependent fashion in a genetically engineered mouse model. These experiments will shed light on a fundamental pathway in heart development that can contribute to the pathogenesis of human disease. We will develop an atlas of let-7 expression and a series of adenoviral and mouse reagents that will be resources for the community's investigation of miRNAs in both developmental and adult contexts. Since miRNAs are amongst the most exciting target molecules of future therapies, elaboration of a let-7/TBX5 regulatory network has the potential to generate major impact on the development of therapeutic strategies for congenital heart disease. Public Health Relevance: This research is devoted to deciphering molecular mechanisms that go awry in birth defects that affect the heart. Approximately 4000 Americans die annually with such heart defects, and identifying specific molecules that regulate the molecular events that cause these disorders will suggest new therapies for these individuals. The types of molecules being studied in this project are ideal candidate targets for such novel approaches to treat individuals with congenital heart defects.